STUDY QUESTION: Is nanopore sequencing for PGT-A analysis of pooled polar bodies a reliable, fast, and cost-effective method and applicable for routine diagnostics in human reproductive care? SUMMARY ANSWER: Nanopore sequencing of pooled polar bodies (PB) revealed high concordance rate with traditional array comparative genomic hybridization (aCGH) analysis and the nanopore sequencing workflow was fast (feasible in under 5 hours) and cost-effective (100-150 Euro per sample), allowing fresh embryo transfer. WHAT IS KNOWN ALREADY: PGT-A using PB biopsy derives a clinical benefit by reducing number of embryo transfers and miscarriage rates but is currently not cost-efficient. Results are often unclear and require expert review. Nanopore sequencing technology opens possibilities by providing cost-efficient, fast sequencing results with uncomplicated sample preparation workflows. Interrogating the polar bodies avoids harming the embryo itself and is the only option for PGT-A in some jurisdictions. STUDY DESIGN, SIZE, DURATION: In this prospective clinical trial, 102 pooled PB samples from 20 patients treated for infertility between March and December 2022 were analyzed for aneuploidy using nanopore sequencing technology and compared with aCGH results generated as part of the clinical routine. All patients participating in this trial were treated for infertility at Wunschbaby Institut Feichtinger (WIF) in Vienna and chose aneuploidy screening of their polar bodies. All patients provided written informed consent. PARTICIPANTS/MATERIALS, SETTING, METHODS: PB samples were analyzed by aCGH for routine PGT-A. Aliquots of whole-genome amplified DNA were anonymized and prepared for sequencing by end-prepping, barcoding and adapter ligation. Samples were pooled equimolar for sequencing on a Nanopore MinION machine. Samples were sequenced for up to 9 hours for 6 pooled PB samples. Whole-chromosome copy-numbers were called by a custom bioinformatic analysis software after alignment and pre-processing. Automatically called results were compared to aCGH results. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 99 pooled polar body samples were compared, three samples were excluded because of failed or uninterpretable results. Overall, the Nanopore sequencing workflow showed high concordance rates with aCGH: 96 of 99 samples were consistently detected as euploid or aneuploid (concordance=97%, specificity = 0.957, sensitivity = 1.0, PPV = 0.906, NPV = 1.0) and 91 samples showed a fully concordant karyotype (92%). Chromosomal aneuploidies analyzed in this trial covered all 23 chromosomes with 98 trisomies, 97 monosomies in 70 aCGH samples. Detailed calculation of time and cost for the nanopore sequencing workflow was performed for different scenarios. Time calculation revealed that the whole nanopore workflow is feasible in under 5 hours (for one sample) with maximum time of 16 hours (for 12 samples in parallel). This enables fresh PB euploid embryo transfer. Material cost for the whole workflow range between 100Euro and 150Euro, including sequencing cost of only 40Euro per sample, resulting in cost-efficient aneuploidy screening. LIMITATIONS, REASONS FOR CAUTION: For some samples, the reference result remained unclear, due to increased noise and limited resolution of the aCGH method. In particular, the small chromosomes show higher variability in both platforms and manual review is often required. A larger study with follow-up data or a clinical non- selection trial would be beneficial for increased confidence. WIDER IMPLICATIONS OF THE FINDINGS: This is the first clinical study, systematically comparing nanopore sequencing for aneuploidy of pooled polar bodies with standard detection methods. High concordance rates confirmed feasibility of nanopore technology for this application. Additionally, the fast and cost-efficient sequencing workflow reveals clinical utility of this technology, making polar body PGT-A clinically attractive. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Wunschbaby Institut Feichtinger, Dr. Wilfried Feichtinger GmbH. The authors declare no competing conflict of interest. TRIAL REGISTRATION NUMBER: Not available